Transient patterns in nematic liquid crystals: Domain-wall dynamics

We study the dynamics of the late stages of the Fréedericksz transition in which a periodic transient pattern decays to a final homogeneous state. A stability analysis of an unstable stationary pattern is presented, and equations for the evolution of the domain walls are obtained. Using results of previous theories, we analyze the effect that the specific dynamics of the problem, incorporating hydrodynamic couplings, has on the expected logarithmic domain growth law.

Mutations in the DNA-binding domain of NR2E3 affect in vivo dimerization and interaction with CRX.

BACKGROUND: NR2E3 (PNR) is an orphan nuclear receptor essential for proper photoreceptor determination and differentiation. In humans, mutations in NR2E3 have been associated with the recessively inherited enhanced short wavelength sensitive (S-) cone syndrome (ESCS) and, more recently, with autosomal dominant retinitis pigmentosa (adRP). NR2E3 acts as a suppressor of the cone generation program in late mitotic retinal progenitor cells. In adult rod photoreceptors, NR2E3 represses cone-specific gene expression and acts in concert with the transcription factors CRX and NRL to activate rod-specific genes. NR2E3 and CRX have been shown to physically interact in vitro through their respective DNA-binding domains (DBD). The DBD also contributes to homo- and heterodimerization of nuclear receptors. METHODOLOGY/PRINCIPAL FINDINGS: We analyzed NR2E3 homodimerization and NR2E3/CRX complex formation in an in vivo situation by Bioluminescence Resonance Energy Transfer (BRET(2)). NR2E3 wild-type protein formed homodimers in transiently transfected HEK293T cells. NR2E3 homodimerization was impaired in presence of disease-causing mutations in the DBD, except for the p.R76Q and p.R104W mutant proteins. Strikingly, the adRP-linked p.G56R mutant protein interacted with CRX with a similar efficiency to that of NR2E3 wild-type and p.R311Q proteins. In contrast, all other NR2E3 DBD-mutant proteins did not interact with CRX. The p.G56R mutant protein was also more effective in abolishing the potentiation of rhodospin gene transactivation by the NR2E3 wild-type protein. In addition, the p.G56R mutant enhanced the transrepression of the M- and S-opsin promoter, while all other NR2E3 DBD-mutants did not. CONCLUSIONS/SIGNIFICANCE: These results suggest different disease mechanisms in adRP- and ESCS-patients carrying NR2E3 mutations. Titration of CRX by the p.G56R mutant protein acting as a repressor in trans may account for the severe clinical phenotype in adRP patients.

The BH4 domain of Bcl-X(L) rescues astrocyte degeneration in amyotrophic lateral sclerosis by modulating intracellular calcium signals.

Collective evidence indicates that motor neuron degeneration in amyotrophic lateral sclerosis (ALS) is non-cell-autonomous and requires the interaction with the neighboring astrocytes. Recently, we reported that a subpopulation of spinal cord astrocytes degenerates in the microenvironment of motor neurons in the hSOD1(G93A) mouse model of ALS. Mechanistic studies in vitro identified a role for the excitatory amino acid glutamate in the gliodegenerative process via the activation of its inositol 1,4,5-triphosphate (IP(3))-generating metabotropic receptor 5 (mGluR5). Since non-physiological formation of IP(3) can prompt IP(3) receptor (IP(3)R)-mediated Ca(2+) release from the intracellular stores and trigger various forms of cell death, here we investigated the intracellular Ca(2+) signaling that occurs downstream of mGluR5 in hSOD1(G93A)-expressing astrocytes. Contrary to wild-type cells, stimulation of mGluR5 causes aberrant and persistent elevations of intracellular Ca(2+) concentrations ([Ca(2+)](i)) in the absence of spontaneous oscillations. The interaction of IP(3)Rs with the anti-apoptotic protein Bcl-X(L) was previously described to prevent cell death by modulating intracellular Ca(2+) signals. In mutant SOD1-expressing astrocytes, we found that the sole BH4 domain of Bcl-X(L), fused to the protein transduction domain of the HIV-1 TAT protein (TAT-BH4), is sufficient to restore sustained Ca(2+) oscillations and cell death resistance. Furthermore, chronic treatment of hSOD1(G93A) mice with the TAT-BH4 peptide reduces focal degeneration of astrocytes, slightly delays the onset of the disease and improves both motor performance and animal lifespan. Our results point at TAT-BH4 as a novel glioprotective agent with a therapeutic potential for ALS.

Identification of SPLUNC1's ENaC-inhibitory domain yields novel strategies to treat sodium hyperabsorption in cystic fibrosis airways.

The epithelial sodium channel (ENaC) is responsible for Na+ and fluid absorption across colon, kidney, and airway epithelia. We have previously identified SPLUNC1 as an autocrine inhibitor of ENaC. We have now located the ENaC inhibitory domain of SPLUNC1 to SPLUNC1's N terminus, and a peptide corresponding to this domain, G22-A39, inhibited ENaC activity to a similar degree as full-length SPLUNC1 (∼2.5 fold). However, G22-A39 had no effect on the structurally related acid-sensing ion channels, indicating specificity for ENaC. G22-A39 preferentially bound to the β-ENaC subunit in a glycosylation-dependent manner. ENaC hyperactivity is contributory to cystic fibrosis (CF) lung disease. Addition of G22-A39 to CF human bronchial epithelial cultures (HBECs) resulted in an increase in airway surface liquid height from 4.2±0.6 to 7.9±0.6 μm, comparable to heights seen in normal HBECs, even in the presence of neutrophil elastase. Our data also indicate that the ENaC inhibitory domain of SPLUNC1 may be cleaved away from the main molecule by neutrophil elastase, which suggests that it may still be active during inflammation or neutrophilia. Furthermore, the robust inhibition of ENaC by the G22-A39 peptide suggests that this peptide may be suitable for treating CF lung disease.

Regulation of P-selectin glycoprotein ligand 1 (PSGL-1) interactions with selectins : role of the decameric repeats and of the cytoplasmic domain

SUMMARY BACKGROUND: P-selectin glycoprotein ligand 1 (PSGL-1) is a major selectin ligand, mediating leukocyte rolling along inflamed vascular wall. It is a mucin-like homodimer composed of a N-terminal domain which binds selectins, followed by 14-16 decameric repeats (DR), a transmembrane domain and a cytoplasmic tail, which may be involved in regulating leukocyte rolling and in generating intracellular signals, through its binding to moesin and Syk. P- and L-selectin binding is dependent on core-2 O-glycosylation and tyrosine sulfation of PSGL-1 N-terminus. However, a minor part of E-selectin-mediated rolling is dependent on N-terminal O-glycans; additional binding sites may thus be involved. In this project, we studied whether (1) PSGL-1 DR and (2) PSGL-1 cytoplasmic residues which bind moesin, were also involved in the regulation of selectin-dependent rolling. METHODS: Several mutated cDNAs were obtained: (1) PSGL-1 DR were either deleted, or substituted by platelet GPlba macroglycopeptide, (2) Ser-336, -348, Lys-337 and Arg-338 were mutated to alanine; moreover, truncation mutants retaining only 6 or 2 cytoplasmic residues were also generated. Transfected CHO expressing mutant PSGL-1 were tested for their ability to bind soluble selectin chimeras and to support selectin-dependent rolling under flow conditions. RESULTS: (1) Deletion of the DR had a dramatic effect on P- and L-selectin-dependent cell recruitment and rolling stability, which could only partially be compensated for, by GPlba substitution. In addition, we observed that DR create a binding site for E-selectin and thus support PSGL-1-dependent rolling. (2) Flow assays revealed that the moesin-binding site, in particular Ser-336, plays a crucial role in regulating the recruitment, velocity and rolling stability of PSGL-1-expressing cells on P- and L-selectin. CONCLUSIONS: Data presented here highlight the structure -function relationship of PSGL-1 DR. Moreover, they reveal a crucial role for the moesin-binding residues in regulating P-and L-selectin-dependent rolling. RÉSUMÉ CONTEXTE: PSGL-1 (P-selectin glycoprotein ligand 1) est un ligand majeur des sélectines permettant le roulement des leucocytes le long de la paroi vasculaire enflammée. C'est un homodimère de type mucine, composé d'un domaine N-terminal liant les sélectines, suivi de 14-16 répétitions décamèriques (RD), d'un domaine transmembranaire et d'une queue cytoplasmique qui pourrait être impliquée dans la régulation du roulement leucocytaire et la génération de signaux intracellulaires, via sa liaison à la moésine et à Syk. La liaison à la Pet à la L-sélectine dépend de la présentation par le N-terminus de PSGL-1 de O-glycans sur des structures core-2 et de tyrosines sulfatées. Cependant, une fraction mineure du roulement médié par la E-sélectine dépend des O-glycans N-terminaux; des sites de liaisons supplémentaires pourraient donc être impliqués. Dans ce projet, nous avons étudié si (1) les RD de PSGL-1 ainsi que (2) les résidus cytoplasmiques liant la moésine, étaient impliqués dans la régulation du roulement dépendant des sélectines. MÉTHODES: Plusieurs ADN codant des formes mutées de PSGL-1 ont été obtenus: (1) Les RD de PSGL-1 ont été soit ôtées, soit remplacées par le macroglycopeptide de la GPlba plaquettaire, (2) les Ser-336, -348, la Lys-337 et l'Arg-338 ont été mutées en alanine; par ailleurs, des mutants tronqués ne retenant plus que 6 ou 2 résidus cytoplasmiques ont également été générés. Des CHO transfectées exprimant PSGL-1 muté ont été testées pour leur capacité à lier des sélectines chimériques solubles et à soutenir un roulement dépendant des sélectines dans des conditions de flux. RÉSULTATS: (1) La perte des RD a eu un effet dramatique sur le recrutement cellulaire et la stabilité de roulement dépendant des P- et L-sélectine, qui n'a pu être que partiellement compensé par la substitution par la GPlba. De plus, nous avons observé que les RD forment un site de liaison pour la E-sélectine et soutiennent ainsi le roulement dépendant de PSGL-1. (2) Les tests de flux ont révélé que le site de liaison à la moésine, notamment la Ser-336, joue un rôle crucial dans la régulation du recrutement, de la vitesse et de la stabilité du roulement des cellules exprimant PSGL-1 sur les P- et L-sélectine. CONCLUSIONS; Les données présentées ici ont permis d'éclaircir la relation structure -fonction des RD de PSGL-1. Par ailleurs, elles révèlent un rôle crucial pour les résidus liant la moésine dans le roulement dépendant des P- et L-sélectine. RÉSUMÉ DESTINÉ À UN LARGE PUBLIC Pour accomplir ses fonctions, le sang circule sur un réseau de 96'000 kilomètres; ainsi, il approvisionne les cellules de l'organisme en énergie, il transporte diverses substances, il assure la défense contre les pathogènes et il participe à la régulation de la température corporelle. Le sang contient plusieurs types de cellules: la grande majorité sont les globules rouges, auxquels il faut ajouter les plaquettes (dont le rôle est de colmater les lésions vasculaires) et les globules blancs (leucocytes) qui, bien que présents en très faible quantité (moins de 0.01 %), jouent un rôle crucial en cas d'infection ou d'inflammation. Une attaque par un pathogène provoque plusieurs changements (rougeur, chaleur, gonflement, douleur), qui sont des manifestations de l'inflammation. Pour atteindre l'agent infectieux, des globules blancs spécialisés (les granulocytes) doivent quitter la circulation sanguine. Afin de faciliter leur capture, les vaisseaux sanguins vont exprimer des protéines telles que les sélectines, qui sont reconnues par une protéine leucocytaire appelée PSGL-1 (P-selectin glycoprotein ligand 7). L'interaction des sélectines avec PSGL-1 soutient le roulement du globule blanc le long de la paroi vasculaire, à une vitesse très inférieure à celle du flux sanguin. Ce roulement conduit à l'activation du globule blanc par des molécules de l'inflammation, permettant son adhésion ferme, puis son arrêt. Finalement, le granulocyte va migrer à travers la paroi du vaisseau pour atteindre et éliminer les causes de l'inflammation. L'adhésion est un processus intéressant à caractériser, car outre l'inflammation, il est également impliqué dans l'artériosclérose, l'infarctus, la métastatisation et la thrombose. Dans ce travail, nous nous sommes intéressés à définir les rôles des différents domaines de PSGL-1 dans la régulation de son interaction avec les sélectines. En effet, en plus de son extrémité extracellulaire de haute affinité pour les sélectines, PSGL-1 est composé de plusieurs séquences répétées hautement glycosylées et d'une courte région intracellulaire, dont les fonctions n'avaient pas été étudiées auparavant. En créant des formes mutées de PSGL-1, nous avons pu montrer qu'un roulement efficace des leucocytes nécessite la présence des régions répétitives et du domaine intracellulaire au complet.

The C-terminal domain of Plasmodium falciparum merozoite surface protein 3 self-assembles into alpha-helical coiled coil tetramer.

Proteins located on the surface of the pathogenic malaria parasite Plasmodium falciparum are objects of intensive studies due to their important role in the invasion of human cells and the accessibility to host antibodies thus making these proteins attractive vaccine candidates. One of these proteins, merozoite surface protein 3 (MSP3) represents a leading component among vaccine candidates; however, little is known about its structure and function. Our biophysical studies suggest that the 40 residue C-terminal domain of MSP3 protein self-assembles into a four-stranded alpha-helical coiled coil structure where alpha-helices are packed "side-by-side". A bioinformatics analysis provides an extended list of known and putative proteins from different species of Plasmodium which have such MSP3-like C-terminal domains. This finding allowed us to extend some conclusions of our studies to a larger group of the malaria surface proteins. Possible structural and functional roles of these highly conserved oligomerization domains in the intact merozoite surface proteins are discussed.

Radiometric age constraints on mineral growth, metamorphism, and tectonism of the Gummfluh klippe, Brianconnais domain of the Prealpes, Switzerland

A combined Ar-40/Ar-39, K/Ar, Rb/Sr and stable isotope study has been made of white micas from the Gummfluh klippe (Brianconnais domain of the Prealpes), Switzerland. The klippe consists mainly of Mesozoic to early Tertiary carbonate rocks metamorphosed from anchizonal to epizonal conditions. At the base of the klippe is a 10-50 m thick, ductilely deformed marble mylonite containing deformed authigenic quartz segregations. Stable isotope measurements of the coexisting calcite (deltaO-18SMOW=24.5) and quartz (deltaO-18SMOW=28.4) from the mylonite indicate relatively low temperatures (< 300-degreesC) during mylonitization. Analyses of white mica separates of varying size fractions from the mylonitic rocks by K/Ar and Rb/Sr techniques yield ages between 57 and 103 Ma. This variation is correlated with two parameters, the size of the mineral fraction, and the proportion of 2M1 (more phengitic) to 1M (more muscovitic) polytype in the sample. The K/Ar and Rb/Sr ages are generally younger in the smaller size fractions, which also containless 2M1 phengite. High precision Ar-40/Ar-39 age spectra from different size fractions of these micas record three distinct components, a small Hercynian component (ca. 200-300 Ma), a significant Eoalpine component (64-80 Ma) forming Ar-40/Ar-39 age plateaus, and a very minor Tertiary component (ca. 20-40 Ma). Characterization of the samples by SEM indicates the presence of two white mica populations, a coarser grained, deformed, detrital mica that probably corresponds to the 2M1 phengite and a finer grained neoformed 1M mica. Collectively these observations suggest that the Gummfluh samples contain a mixture of detrital phengites of Hercynian age together with neocrystallized muscovites grown during the late Eoalpine metamorphic event followed by minor argon loss during the Tertiary. The main geologic episode recorded in the Ar-40/Ar-39 age spectra of white micas in the mylonite is of Late Cretaceous/Early Tertiary age (64-80 Ma), representing the first reliable Eoalpine ages ever to be reported from the Prealpes. Contrary to tectonic models, the marble mylonite at the base of the Gummfluh klippe appears to be a Cretaceous thrust plane and not the thrust surface formed during transport of the klippe into its present position from the Penninic Alps during the Tertiary. The late Cretaceous thrust developed during marine sedimentation at a depth of 800 m below the seafloor at temperatures of approximately 280-degrees-C, facilitated by warm fluids along the tectonic discontinuity.

Coexpression of the T-cell receptor constant alpha domain triggers tumor reactivity of single-chain TCR-transduced human T cells.

Transfer of tumor antigen-specific T-cell receptors (TCRs) into human T cells aims at redirecting their cytotoxicity toward tumors. Efficacy and safety may be affected by pairing of natural and introduced TCRalpha/beta chains potentially leading to autoimmunity. We hypothesized that a novel single-chain (sc)TCR framework relying on the coexpression of the TCRalpha constant alpha (Calpha) domain would prevent undesired pairing while preserving structural and functional similarity to a fully assembled double-chain (dc)TCR/CD3 complex. We confirmed this hypothesis for a murine p53-specific scTCR. Substantial effector function was observed only in the presence of a murine Calpha domain preceded by a TCRalpha signal peptide for shuttling to the cell membrane. The generalization to a human gp100-specific TCR required the murinization of both C domains. Structural and functional T-cell avidities of an accessory disulfide-linked scTCR gp100/Calpha were higher than those of a dcTCR. Antigen-dependent phosphorylation of the proximal effector zeta-chain-associated protein kinase 70 at tyrosine 319 was not impaired, reflecting its molecular integrity in signaling. In melanoma-engrafted nonobese diabetic/severe combined immunodeficient mice, adoptive transfer of scTCR gp100/Calpha transduced T cells conferred superior delay in tumor growth among primary and long-term secondary tumor challenges. We conclude that the novel scTCR constitutes a reliable means to immunotherapeutically target hematologic malignancies.

Trypsin cleaves acid-sensing ion channel 1a in a domain that is critical for channel gating.

Acid-sensing ion channels (ASICs) are neuronal Na(+) channels that are members of the epithelial Na(+) channel/degenerin family and are transiently activated by extracellular acidification. ASICs in the central nervous system have a modulatory role in synaptic transmission and are involved in cell injury induced by acidosis. We have recently demonstrated that ASIC function is regulated by serine proteases. We provide here evidence that this regulation of ASIC function is tightly linked to channel cleavage. Trypsin cleaves ASIC1a with a similar time course as it changes ASIC1a function, whereas ASIC1b, whose function is not modified by trypsin, is not cleaved. Trypsin cleaves ASIC1a at Arg-145, in the N-terminal part of the extracellular loop, between a highly conserved sequence and a sequence that is critical for ASIC1a inhibition by the venom of the tarantula Psalmopoeus cambridgei. This channel domain controls the inactivation kinetics and co-determines the pH dependence of ASIC gating. It undergoes a conformational change during inactivation, which renders the cleavage site inaccessible to trypsin in inactivated channels.

Crystallization and preliminary X-ray diffraction studies of Drosophila melanogaster Gαo-subunit of heterotrimeric G protein in complex with the RGS domain of CG5036.

Regulator of G-protein signalling (RGS) proteins negatively regulate heterotrimeric G-protein signalling through their conserved RGS domains. RGS domains act as GTPase-activating proteins, accelerating the GTP hydrolysis rate of the activated form of Gα-subunits. Although omnipresent in eukaryotes, RGS proteins have not been adequately analysed in non-mammalian organisms. The Drosophila melanogaster Gαo-subunit and the RGS domain of its interacting partner CG5036 have been overproduced and purified; the crystallization of the complex of the two proteins using PEG 4000 as a crystallizing agent and preliminary X-ray crystallographic analysis are reported. Diffraction data were collected to 2.0 Å resolution using a synchrotron-radiation source.

Analysis of the Cytoprotective Role of α-Crystallins in Cell Survival and Implication of the αA-Crystallin C-Terminal Extension Domain in Preventing Bax-Induced Apoptosis.

α-Crystallins, initially described as the major structural proteins of the lens, belong to the small heat shock protein family. Apart from their function as chaperones, α-crystallins are involved in the regulation of intracellular apoptotic signals. αA- and αB-crystallins have been shown to interfere with the mitochondrial apoptotic pathway triggering Bax pro-apoptotic activity and downstream activation of effector caspases. Differential regulation of α-crystallins has been observed in several eye diseases such as age-related macular degeneration and stress-induced and inherited retinal degenerations. Although the function of α-crystallins in healthy and diseased retina remains poorly understood, their altered expression in pathological conditions argue in favor of a role in cellular defensive response. In the Rpe65(-/-) mouse model of Leber's congenital amaurosis, we previously observed decreased expression of αA- and αB-crystallins during disease progression, which was correlated with Bax pro-death activity and photoreceptor apoptosis. In the present study, we demonstrated that α-crystallins interacted with pro-apoptotic Bax and displayed cytoprotective action against Bax-triggered apoptosis, as assessed by TUNEL and caspase assays. We further observed in staurosporine-treated photoreceptor-like 661W cells stably overexpressing αA- or αB-crystallin that Bax-dependent apoptosis and caspase activation were inhibited. Finally, we reported that the C-terminal extension domain of αA-crystallin was sufficient to provide protection against Bax-triggered apoptosis. Altogether, these data suggest that α-crystallins interfere with Bax-induced apoptosis in several cell types, including the cone-derived 661W cells. They further suggest that αA-crystallin-derived peptides might be sufficient to promote cytoprotective action in response to apoptotic cell death.

An SH2 domain-containing 5' inositolphosphatase inhibits insulin-induced GLUT4 translocation and growth factor-induced actin filament rearrangement.

Tyrosine kinase receptors lead to rapid activation of phosphatidylinositol 3-kinase (PI3 kinase) and the subsequent formation of phosphatidylinositides (PtdIns) 3,4-P2 and PtdIns 3,4, 5-P3, which are thought to be involved in signaling for glucose transporter GLUT4 translocation, cytoskeletal rearrangement, and DNA synthesis. However, the specific role of each of these PtdIns in insulin and growth factor signaling is still mainly unknown. Therefore, we assessed, in the current study, the effect of SH2-containing inositol phosphatase (SHIP) expression on these biological effects. SHIP is a 5' phosphatase that decreases the intracellular levels of PtdIns 3,4,5-P3. Expression of SHIP after nuclear microinjection in 3T3-L1 adipocytes inhibited insulin-induced GLUT4 translocation by 100 +/- 21% (mean +/- the standard error) at submaximal (3 ng/ml) and 64 +/- 5% at maximal (10 ng/ml) insulin concentrations (P < 0.05 and P < 0.001, respectively). A catalytically inactive mutant of SHIP had no effect on insulin-induced GLUT4 translocation. Furthermore, SHIP also abolished GLUT4 translocation induced by a membrane-targeted catalytic subunit of PI3 kinase. In addition, insulin-, insulin-like growth factor I (IGF-I)-, and platelet-derived growth factor-induced cytoskeletal rearrangement, i.e., membrane ruffling, was significantly inhibited (78 +/- 10, 64 +/- 3, and 62 +/- 5%, respectively; P < 0.05 for all) in 3T3-L1 adipocytes. In a rat fibroblast cell line overexpressing the human insulin receptor (HIRc-B), SHIP inhibited membrane ruffling induced by insulin and IGF-I by 76 +/- 3% (P < 0.001) and 68 +/- 5% (P < 0.005), respectively. However, growth factor-induced stress fiber breakdown was not affected by SHIP expression. Finally, SHIP decreased significantly growth factor-induced mitogen-activated protein kinase activation and DNA synthesis. Expression of the catalytically inactive mutant had no effect on these cellular responses. In summary, our results show that expression of SHIP inhibits insulin-induced GLUT4 translocation, growth factor-induced membrane ruffling, and DNA synthesis, indicating that PtdIns 3,4,5-P3 is the key phospholipid product mediating these biological actions.

Long-lived States in an intrinsically disordered protein domain

Long-lived states (LLS) are relaxation-favoured eigenstates of J-coupled magnetic nuclei. LLS were measured, along with classical 1H and 15 N relaxation rate constants, in aminoacids of the N-terminal Unique domain of the c-Src kinase (USrc), which is disordered in vitro under physiological conditions. The relaxation rates of LLS are a probe for motions and interactions in biomolecules. LLS of the aliphatic protons of glycines, with lifetimes ca. four times longer than their spin-lattice relaxation times, are reported for the first time in an intrinsically disordered protein domain (IDP). LLS relaxation experiments were integrated with 2D spectroscopy methods, further adapting them for studies on proteins.